WO2020176815A2 - Procédé à haut rendement basé sur une séquence générant des anticorps de camélidé pour couvrir de larges épitopes avec une haute résolution - Google Patents

Procédé à haut rendement basé sur une séquence générant des anticorps de camélidé pour couvrir de larges épitopes avec une haute résolution Download PDF

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WO2020176815A2
WO2020176815A2 PCT/US2020/020248 US2020020248W WO2020176815A2 WO 2020176815 A2 WO2020176815 A2 WO 2020176815A2 US 2020020248 W US2020020248 W US 2020020248W WO 2020176815 A2 WO2020176815 A2 WO 2020176815A2
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vhh
antibody
sequences
antigen
lineage
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WO2020176815A3 (fr
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Weimin Zhu
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Zhejiang Nanomab Technology Center Co. Ltd.
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Priority to CN202080031628.1A priority Critical patent/CN114126646A/zh
Priority to JP2021549905A priority patent/JP2022541697A/ja
Priority to EP20763951.9A priority patent/EP3930754A4/fr
Priority to US17/434,722 priority patent/US20230002757A1/en
Publication of WO2020176815A2 publication Critical patent/WO2020176815A2/fr
Publication of WO2020176815A3 publication Critical patent/WO2020176815A3/fr

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Definitions

  • Targeting functional epitopes of a disease target for therapeutics is a big challenge with current antibody technologies because each target has hundreds or thousands of epitopes and only a very limited number of epitopes among them are involved in the biological function, but current technologies are taking approaches to generate binders randomly and sporadically, thus, inadequate coverage of epitopes, redundant selection and low successful rate are the bottle-neck.
  • Camelus dromedaries, bactrianus belong to old world Camelidae and Lama,
  • HcAbs have evolved comprehensive paratope architecture as one of the driving factors for recognizing the very wide range of epitopes of the antigen, and IgGl antibodies complement HcAb binding architecture for more diverse recognitions.
  • Camelidae have unique humoral immune system consisting of 2 types of HcAb, lgG2 and lgG3 with short and long length of hinge regions.
  • Phylogenetic analyses have confirmed that HcAbs diverged from a conventional antibody, IgGl as a result of recent adaptive changes. It was reported that IgGl and lgG3 neutralize West Nile virus, whereas lgG2 seems less effective in an infected or vaccinated animal (Daley LP, Clin. Vaccine Immunol.
  • HcAb and IgGl can overlap, but HcAb can also reach sites inaccessible to IgGl. Understanding of the exact roles and functions of the various Camelids IgG isotypes is still in its infancy.
  • the diverse paratope architecture such as prolate, convex, concave, protrude, and flat surfaces of HcAb (lgG2 and lgG3) offer a great opportunity to develop antibody to challenging targets, especially for diagnostic and therapeutic applications.
  • the simplicity of HcAb without light chain pairing also makes gene cloning and antibody engineering much easier.
  • the conventional IgGl contributing to 25-50% of total IgG of Camelids play important role to expand an antigen binding repertoire since the HcAb repertoire of an immunized dromedary or llama displays a recognition pattern different from that of conventional IgGl (McCoy LE, J. Exp. Med. 2012), and certain unique epitopes or druggable target hotspots are accessible to IgGl with high affinity and desired functionality (Cristina Basilico, The Journal of Clinical Investigation, Volume 124 Number 7 July, 2014; Basvan der Woninga, MABS, VOL. 8, NO. 6, 1126-1135, 2016).
  • VK VX pairing with VH 1 to form conventional IgGl
  • their germline organizations have been revealed recently (Laura M. Griffin, Journal of Immunological Methods Volume 405, Pages 35-46, March 2014; Alex Klarenbeek, mAbs 7:4, 693-706; 2015).
  • VHH domain of HcAb enlarges overall antigen-binding repertoire, for example by creating prolate (rugby ball-shaped) structure with a convex paratope surface, which makes it extremely suitable to insert in cavities or clefts (such as active and allosteric sites) on the surface of the antigen.
  • the VH-VL domain of conventional IgG contains more flat or concave paratope surface.
  • VHH contains the Framework Region 2 (FR2) with hydrophilic amino acid substitutions comparing to conventional FR2 (Val37 -> Phe/Tyr, Gly44 -> Glu, Leu45 -> Arg, and Trp47 -> Gly), which participates in the light chain binding
  • FR2 Framework Region 2
  • extended CDR1 region with extensive somatic hypermutation in immune B-cells in residues 27-30 according to Kabat's numbering
  • extra disulfide bonds within CDR1 and CDR3 in certain portion of VHH (v) longer CDR3 loop is also identified possibly due to additional non-templated nucleotide insertions in some VHH (Adhdi Arbabi-Ghahroudi, Frontiers in Immunology, Vol 8, 2017; Vie
  • VHH non-classical VHH (without FR2 hydrophilic amino acids) which are derived from the same gene locus, IGHV3 or IGHV4, D and J as conventional IgGl do, and these heavy chain antibodies may recognize the same or similar epitopes as IgGl since both category antibodies share the same or similar CDR3 which is responsible for epitope recognition (Conrath KE Dev Comp Immunol 27:87-103, 2003; Nick Deschacht, The Journal of Immunology. 184 (10) 5696-5704, 2010).
  • the HcAb germline organization and VHH structure are illustrated in Fig 1. A and B.
  • This invention disclosed a high-throughput method generating a camelid antibody against an antigen, comprising: a) enriching and proliferating B-cells from immunized camelids specific to the antigen, b) generating antibody Next Generation Sequencing (NGS) libraries comprising VHH 2 , VHH 3 , and VH 1 chain sequences from the antigen-specific B-cells, c) grouping sequences of VHH 2 , VHH 3 , and VH 1 in the NGS libraries by lineages, d) ranking the lineages comprising the VHH heavy chain (VHH 2 , VHH 3 ) by one or more lineage priority factors, e) selecting a representative sequence from lineages of VHH heavy chain (VHH 2 and VHH 3 ) with a top ranking in the NGS database library, and f) testing an antibody comprising the selected VHH heavy chain sequences to determine if the antibody binds to the antigen or portion thereof.
  • the antigen comprises a plurality of epitop
  • minimal CDR3 distance of a specific CDR3 is equal or less than 1 among the group of CDR3s from a lineage, wherein minimal CDR3 distance of a specific CDR3 is the smallest hamming distance of this CDR3 comparing with all other CDR3 of the same length.
  • the lineage priority factors are selected from a group consisting of lineages from high to low sequences abundancy, lineages from high to low amplification factor after in vitro B-cells enrichment and proliferation, lineages sequences abundancy change during immunization course, lineages sequences abundancy change before and after depleting certain unwanted B cells, lineages which share the same naive B-cell origin between VHH and VH, avoidance of developability liability sequences, and a combination thereof.
  • VHH 2 and/or VHH 3 lineages are chosen from the top 100 lineages in e).
  • the method further comprises repeating e)-f) to generate camelid antibodies, wherein the representative sequences are selected from top 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400, 1401-1500, 1501-1600, 1601-1700, 1701-1800, 1801-1900, or 1901- 2000 ranking lineages.
  • the method further comprises repeating e)-f) to generate camelid antibodies, wherein the representative sequences are selected from top 2,000 to 10,000 ranking lineages.
  • the testing antibody is expressed by prokaryotic or eukaryotic cells.
  • the method further comprises monitoring immune responses of lgG2, 3 (HcAb) and IgGl (conventional IgG).
  • sequences within the same lineage group of the selected lgG2 or lgG3 heavy chain only antibodies can be chosen for optimization of the antibodies by repeating e)-f) of the method.
  • the antigen or immunogen can be cells, a tissue, or a biofluid.
  • the antigen can be a complex immunogen
  • the method further include: using an antibody which has been determined in step (f) to bind with the complex immunogen to identify individual antigens included in the complex immunogen by protein array, cells/tissue antigen cDNA library, or mass spectrometry-based
  • the method further comprises lineage
  • VHH subgrouping with certain VHH features selected from a group consisting of i) FR2 hydrophilic region; ii) extended CDR1; iii) extra disulfide bond between CDR1-CDR3 or FR2-CDR3; iv) extra disulfide bond within CDR3; v) long CDR3 (>15 aa); vi) extra disulfide bond within CDR1; vii) Non-classic VHH which have the same V and J germlines as conventional IgGl; viii) Non-classic VHH which have certain predetermined sequence signatures; ix) certain predetermined canonical binding loop structure; x) convergent motif or sequence signature among individual animals from the same immunization group; xi) CDR2 length; xii) CDR3 length; xiii) CDR3 length and identity; xiv) presence of 3 or more positive charges in CDR3 region; xv) the number of cysteines in the amino acid sequence; and
  • a method for high-throughput generating a camelid antibody against an antigen comprising: a) enriching and proliferating antigen-specific B-cells from immunized camelids; b) generating antibody NGS libraries comprising VHH 2 , VHH 3 , VH 1 and VL 1 chain sequences from said antigen-specific B-cells; c) grouping VHH 2 , VHH 3 , VH 1 and VL 1 NGS sequences by lineages; d) VHVVL 1 lineage pairing according to an anchor binder generated by single B-cell sorting and heterohybridoma approaches; e) ranking lineages and lineage pairs from step c) and step d) by lineage priority factors; f) selecting a representative sequence or sequence pair from lineages of VHH 2 , VHH 3 and lineage pairs of VHVVL 1 with the top ranking in the NGS library; g) testing an antibody comprising the selected heavy chain/
  • minimal CDR3 distance of a specific CDR3 is equal or less than 1 among the group of CDR3s from a lineage, wherein minimal CDR3 distance of a specific CDR3 is the smallest hamming distance of this CDR3 comparing with all other CDR3 of the same length.
  • the ranking of lineage pairs in step e) is based on lineage priority factors of VH 1 lineages of said lineage pairs.
  • the lineage priority factors are selected from a group consisting of lineages from high to low sequences abundancy, lineages from high to low amplification factor after in vitro B-cells enrichment and proliferation, lineages sequences abundancy change during immunization course, lineages sequences abundancy change before and after depleting certain unwanted B cells, lineages which share the same naive B-cell origin between VHH and VH, avoidance of developability liability sequences, and a combination thereof.
  • anchors for IgGl repertoire are generated with single B- cell sorting and heterohybridoma approaches.
  • testing antibody is expressed by prokaryotic or eukaryotic cells.
  • representative pair VH VVL 1 from each top 100 lineages or lineage pairs are selected.
  • 100 lineages/lineage-pairs include 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10 VHHs lineage and 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 VHVVL 1 lineage pairs respectively, wherein VL 1 comprises VK and VX.
  • the method further comprises repeating f)-g) to generate camelid antibodies, wherein the representative sequences are selected from top 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400, 1401-1500, 1501-1600, 1601-1700, 1701-1800, 1801-1900, or 1901- 2000 ranking lineages.
  • the method further comprises repeating f)-g) to generate camelid antibodies, wherein the representative sequences are selected from top 2000 to 10,000 ranking lineages.
  • the criteria for lineage ranking/selection are selected from a group consisting of lineages from high to low sequences abundancy, lineages from high to low amplification factor, lineages sequences abundancy change during immunization course, lineages sequences abundancy change before and after depleting certain unwanted B cells, Lineages which share the same naive B-cell origin between VHH and VH 1 , avoidance of developability liability sequences, and a combination thereof.
  • the antigen or immunogen can be cells or tissue, and generated VHHs are used to identify individual corresponding antigens by protein array or cells/tissue antigen cDNA library or immunoprecipitation -based mass spectrometry method.
  • the method for high-throughput generating camelid antibody against a plurality of epitopes of a specific antigen further comprises subgrouping VHHs lineages having a feature selected from a group consisting of i) FR2 hydrophilic region; ii) extended CDR1; iii) extra disulfide bond within CDR1 or/and CDR3; iv) long CDR3 (>15 aa); v) sequences sharing the same naive B cells origin among VHH 2 , VHH 3 and VH 1 ; vi) sequence-based prediction for antigen-binding loops structure; x) convergent motif or sequence signature among individual animals from the same immunization group; xi) CDR2 length; xii) CDR3 length; xiii) CDR3 length and identity; xiv) presence of 3 or more positive charges in CDR3 region; xv) the number of cysteines in the amino acid sequence; and
  • sequences within the same lineage group of tested antibodies in the first round can be chosen for optimization of the antibodies by repeating f)-g) in the second round.
  • the method for high-throughput generating camelid antibodies against an antigen further comprises applying the selected VHH sequences to guide VH 1 -VL 1 pair selection for these clones which share the same naive B-cell origin, wherein the selection criteria comprise 1) CDR1 and CDR2 differences; 2) FR1,2,3 and 4 differences.
  • a method for generating humanized VHH antibody comprises a) enriching and proliferating antigen-specific B-cells from immunized camelids; b) generating antibody NGS libraries comprising VHH 2 , VHH 3 , and VH 1 chain sequences from antigen-specific B-cells; c) grouping VHH 2 , VHH 3 , and VH 1 NGS sequences by lineages; d) identifying a substitutable position in a parent VHH 2 , VHH 3 antibody or VH 1 sharing the same naive B-cell origin by comparing its amino acid sequence to the amino acid sequences of a number of related antibodies that each bind to the same epitope as the parent antibody in the same lineage; e) substituting amino acids at one or more of the substitutable positions of the parental VHH 2 or VHH 3 antibody by correspondingly positioned amino acids in the human antibody; f) testing an antibody comprising the substituted residues within the selected sequences to determine if
  • the substituted position of humanized VHH antibody is in the FRs regions. In one embodiment, the substituted position of humanized VHH antibody is in the CDRs regions.
  • the parent antibody is a camelid antibody. In one embodiment, the parent antibody is a humanized camelid antibody.
  • an isolated camelid antibody or antigen binding portion comprising an antibody sequencence generated by this invention.
  • a pharmaceutical composition comprising the camelid antibody of this invention and a pharmaceutically acceptable carrier.
  • Figure 1A shows the organization of camelids locus that encode VHH, VH and CH immunoglobulins.
  • Figure IB shows camelids VHH structure.
  • Figure 2 shows Camelids immunization and antigen-specific B cells enrichment / proliferation.
  • Figure 3 shows IgG isotype specific primer sets to amplify VHH 2 , VHH 3 , VH 1 , Vk and V .
  • Figure 4 shows generating VHH 2 , VHH 3 , VH 1 , Vk and VX NGS libraries and grouping lineages.
  • Figure 5 shows further grouping VHH 2 and VHH 3 lineages with sequence signatures.
  • Figure 6 shows pairing VH-Vk or VH- /l lineages with anchors developed with single B-cells sorting and heterohybridoma.
  • Figure 7 shows sequences selection from each lineage for binder and bioactivity screening.
  • Figure 8 shows optimization of the selected lead within the same lineage. Leads from the first round of selection are marked with @ and +.
  • Figure 9 shows workflow for humanization of VHH and VH-VL through lineage analysis.
  • Figure 10 shows the fluorescence signal from selected antibodies binding to
  • Figure 11 shows ELISA assays of antibodies secreted from selected co-cultures of anti-KLH antibody-secreting B cells and alpaca feeder cells.
  • the B-cells only without the feeder cells will not grow or amplify. It was used as a negative control.
  • the immunized animal serum was diluted 1:1000 with the control media as a positive control for ELISA.
  • Figure 12 shows the amplification of B cells obtained from co-cultures of feeder cells and antibody secreting B cells vs. B-cell only without feeder cells.
  • Figure 13 shows results of ELISA assay of supernatants of selected clones from
  • Figure 14 shows workflow for identification of blocking antibodies based on CDR region 2-4 amino acid motif identified from 3-d structure of ligand/receptor complex.
  • Figure 15 shows two views of the complex formed between PD-1 and PD-L1 highlighting two peptides of PD-1 at the interface of the complex.
  • Figure 16 shows analysis of the interaction of interfacial peptides in a PD-1:PD-L1 complex.
  • Figure 17 shows a phylogenetic tree illustrating lineage grouping of selected anti-
  • Figure 18 shows a Venn diagram illustrating the overlap in CDR3 domain amino acid sequences in clone libraries from immunized alpacas A1 and A2.
  • Figure 19A shows a selection scheme to identify overlapping clones from alpacas
  • Figure 19B shows a Venn diagram illustrating the overlap in hinge region amino acid sequences in clone libraries from immunized alpacas A1 and A2.
  • Figure 20A shows a selection scheme to identify overlapping clones from alpacas
  • Figure 20B shows a Venn diagram illustrating the overlap in hinge region amino acid sequences in clone libraries from immunized alpacas A1 and A2. $$
  • Figure 21A shows a Venn diagram illustrating the overlapping clones of VH, VHH2 and VHH3 antibodies in a library from a single alpaca that have common CDR3 domain sequences.
  • Figure 21B shows the CDR3 sequences in common.
  • Figure 22 is a Venn diagram showing the number of CDR3 domain sequences in common among alpacas A1 and A2, VH, VHH2 and VHH3 antibodies.
  • Figure 23 shows the antigen-binding affinity by FACS using RPMI8226 cells expressing BCMA of selected antibody clones of classical VHH type and non-classical VHH type.
  • Figure 24 shows the proportion of unique FR2 domain sequences in each of three libraries of antibodies binding to three different antigens.
  • Figure 25 shows the frequency of certain amino acid substitutions in the FR2 domain of antibodies in each of three libraries of antibodies binding to three different antigens.
  • Figure 26 shows the proportion of clones having a CDR1 domain length from 11 to 15 amino acids in each of three libraries of antibodies binding to three different antigens.
  • Figure 27 shows the distribution of binding affinity by ELISA of antibodies having
  • Figure 28 shows the proportion of clones having extra disulfide bonds within the
  • Figure 29 and Figure 30 show the proportion of clones having extra disulfide bonds between the CDR1 and CDR2 domains in antibodies from three targeting three different antigens.
  • Figure 31 shows analysis of the number of cysteine residues in the amino acid sequence of V-region in antibodies from three libraries targeting three different antigens.
  • Figure 32 shows the proportion of clones having extra disulfide bonds between the CDR1 and CDR3 domains in antibodies from three libraries targeting three different antigens.
  • Figure 33 shows the proportion of clones having extra disulfide bonds between the FR2 and CDR2 domains or between the FR2 and CDR3 domains in antibodies from three libraries targeting three different antigens.
  • Figure 34 shows the correlation between the number of cysteine amino acids in a VHH antibody sequence and the supernatant OD value.
  • Figure 35 shows the proportion of clones having a "long CDR3" domain in each of three libraries of antibodies binding to three different antigens.
  • Figure 36, Figure 37 and Figure 38 show the correlation between length of the
  • Figure 39 shows the range of CDR3 length for a population of VHH antibodies binding to nearly identical epitopes or the same epitope of a certain antigen.
  • Figure 40 shows the results of an experiment assessing competition of selected anti-PDl clones with KEYTRUDA and OPDIVO for binding to PD1.
  • Figure 41 shows the proportion of clones having the Trpll8 amino acid substituted with Arg in VHH antibodies in each of three libraries of antibodies binding to three different antigens.
  • Figure 42 shows the positive correlation between length of the CDR3 and ELISA binding activity for anti-KLH VHH antibodies.
  • plurality refers to more than 1 , for example more than 2 , more than about 5 , more than about 10 , more than about 20 , more than about 50 , more than about 100 , more than about 200 , more than about 500 , more than about 1000 , more than about 2000 , more than about 5000 , more than about 10 , 000 , more than about 20 , 000 , more than about 50 , 000 , more than about 100 , 000 , usually no more than about 200 , 000 .
  • a "population" contains a plurality of items.
  • the term "about” refers to a measurable value such as an amount, a time duration, and the like, and encompasses variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5% or ⁇ 0.1% from the specified value.
  • epitopic determinants can include any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the equilibrium dissociation constant is ⁇ 1 mM, preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • K D can refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
  • immune response can refer to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from an organism of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal organismal cells or tissues.
  • an "antigen-specific T cell response" as used herein can refer to responses by a T cell that result from stimulation of the T cell with the antigen for which the T cell is specific.
  • responses by a T cell upon antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production).
  • antibody refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region, referred to herein as the "Fc fragment” or "Fc region".
  • Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single region antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • the Fc region includes portions of two heavy chains contributing to two or three classes of the antibody.
  • the Fc region may be produced by recombinant DNA techniques or by enzymatic (e.g. papain cleavage) or via chemical cleavage of intact antibodies.
  • antibody fragment refers to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
  • antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI regions; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI region; (iii) the Fd fragment having VH and CHI regions; (iv) the Fd' fragment having VH and CHI regions and one or more cysteine residues at the C-terminus of the CHI region; (v) the Fv fragment having the VL and VH regions of a single arm of an antibody;
  • polypeptide chain see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.
  • scFv antibodies as used herein refers to forms of antibodies comprising the variable regions of only the heavy (VH) and light (VL) chains, connected by a linker peptide.
  • the scFvs are capable of being expressed as a single chain polypeptide.
  • the scFvs retain the specificity of the intact antibody from which it is derived.
  • the light and heavy chains may be in any order, for example, VH-linker-VL or VL-linker-VH, so long as the specificity of the scFv to the target antigen is retained.
  • an "isolated antibody”, as used herein, can refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a TRAIL protein can be substantially free of antibodies that specifically bind antigens other than TRAIL proteins).
  • An isolated antibody that specifically binds a human TRAIL protein can, however, have cross-reactivity to other antigens, such as TRAIL proteins from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein can refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • recombinant human antibody can refer to all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the term "isotype" can refer to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
  • An antibody can be an immunoglobulin G (IgG), an IgM, an IgE, an IgA or an IgD molecule, or is derived therefrom.
  • VHH 2 is representing the heavy chains of three camelid IgG isotypes lgG2, IgGB and IgGl respectively.
  • VL 1 is representing the light chain of camelid IgGl.
  • Camelid VL 1 includes, but not limited to VK and VX.
  • correspondingly positioned amino acids and “corresponding amino acids” used herein interchangeably, are amino acid residues that are at an identical position (i.e., they lie across from each other) When two or more amino acid sequences are aligned. Methods for aligning and numbering antibody sequences are set forth in great detail in Chothia, supra, Kabat supra, and others. As is known in the art (see, e.g. Kabat 1991 Sequences of Proteins of Immunological Interest, DHHS, Washington, DC), sometimes one, two or three gaps and/or insertions of up to one, two, three or four residues, or up to about 15 residues
  • natural antibody refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multicellular organism.
  • Spleen, lymph nodes, bone marrow, blood and other lymphatic tissues are examples of tissues that contain cells that produce natural antibodies.
  • the antibodies produced by B cells isolated from a first animal immunized with an antigen are natural antibodies.
  • Natural antibodies contain naturally - paired heavy and light chains.
  • naturally paired refers to heavy and light chain sequences that have been paired by the immune system of a multi-cellular organism.
  • mixture refers to a combination of elements, e. g., cells, that are interspersed and not in any particular order.
  • a mixture is homogeneous and not spatially separated into its different constituents.
  • Examples of mixtures of elements include a number of different cells that are present in the same aqueous solution in a spatially undressed manner.
  • assessing includes any form of measurement, and includes determining if an element is present or not.
  • determining includes determining if an element is present or not.
  • evaluating means assessing, assessing and “assaying” are used interchangeably and may include quantitative and / or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, and / or determining whether it is present or absent.
  • enriched is intended to refer to component of a composition (e.g., a particular type of cells ) that is more concentrated (e.g., at least 2x , at least 5x , at least lOx , at least 50x , at least lOOx , at least 500x , at least 1 , OOOx ) , relative to other components in the sample ( e.g., other cells ) than prior to enrichment .
  • a composition e.g., a particular type of cells
  • more concentrated e.g., at least 2x , at least 5x , at least lOx , at least 50x , at least lOOx , at least 500x , at least 1 , OOOx
  • something that is enriched may represent a significant percent (e.g., greater than 2 %, greater than 5 %, greater than 10 %, greater than 20 %, greater than 50 %, or more , usually up to about 90 % - 100 %) of the sample in which it resides .
  • enriching is intended to any way by which antigen - specific cells can be obtained from a larger population of B cells. As described in greater detail below, enriching may be done by panning, using a bead or cell sorting, for example.
  • obtaining in the context of obtaining an element, e. g., cells or sequences, is intended to include receiving the element as well as physically producing the element.
  • PBMCs peripheral blood mononucleated cells
  • T cells, B cells and NK cells lymphocytes
  • monocytes monocytes
  • macrophage macrophage
  • antigen-specific B cells refers to memory B cells that have an antibody that specifically binds to an antigen on their surface, as well as progenitors thereof.
  • a cell is "derived from” a host if the cell, or the progeny thereof, was obtained from the host.
  • the progeny of a progenitor cell is derived from the progenitor cell.
  • the term "support comprising the antigen” comprises any type of support (e.g., a solid or semi - solid support, including plates and beads) that contains an antigen, or a portion thereof, immobilized thereon.
  • An antigen may be immobilized on a support directly or indirectly, e.g., via a linker, via a biotin - streptavidin interaction or via a cell, for example.
  • the term "panning” is used to refer to a method by which B cells are applied to a container (e.g., a plate) that has one or more surfaces that are coated in an antigen or portion thereof. Unbound cells can be removed by washing the surface after the cells are applied to it.
  • beads - based enrichment is used to refer to a method by which B cells are mixed with beads, e.g., magnetic beads, that are linked to an antigen or portion thereof.
  • cell sorting is used to refer to a method by which B cells are mixed a detectable antigen (e.g., a fluorescently detectable antigen) in solution.
  • a detectable antigen e.g., a fluorescently detectable antigen
  • FACS Fluorescence - activated cell sorting
  • complex immunogen is intended to refer to an immunogen that contains a plurality of antigens.
  • a complex immunogen can be composed of a plurality of different antigens that have been separately made and then mixed together, or they may be naturally complex (e.g., as is the case when one uses an entire cell and tissue or a fraction thereof) in an immunization.
  • the term "activating" is referred to the stimulation of B cells to a) proliferate and b) differentiate into plasma blasts and / or plasma cells and c) secrete antibodies.
  • B cell activation can be done by contacting the B cells with antigen, T cells expressing CD40L and cytokines, although other methods are known (see, e.g., Wykes, Imm. Cell. Biol. 2003 81: 328 - 331).
  • activated B cells refers to a cell population that comprises the progeny of a B cell that was activated. As noted above, activation causes B cells to proliferate, and the progeny of such cells are referred to herein as activated B cells.
  • collecting refers to the act of separating the cells that in the culture medium from a substrate. Collecting may be done by pipetting or by decanting, for example.
  • immunized animal is intended to refer to any animal (humans, rabbits, mice , rats , sheep , cows , chickens , camels ) that is mounting an immune response an antigen .
  • An animal may be exposed to a foreign antigen via exposure to an infectious agent, a vaccination, or by administrating an antigen and adjuvant (e.g., by injection), for example.
  • the term "immunized by an antigen” is also intended to include animals that are mounting an immune response against a "self” antigen, i.e., have an autoimmune disease.
  • ranking and “ranked order of abundance” refer to the order of sequences when they are listed by their abundance, i.e., with the most abundant sequence first, the second most abundant sequence next, and the third most abundant sequence next, and so on. In certain cases, sequences may be ranked by making a frequency distribution, and then ordering the sequences by their frequency.
  • corresponding rank or “correspondingly ranked” refer to two sequences that have the same positions in two ranks.
  • first, second and third positions in a first rank correspond to the first, second and third positions in a second rank, respectively.
  • lineage rank refers to the order of lineages when they are listed by their priority factors.
  • the priority factors include but not limited to abundancy of lineage sequences, amplification factor, dynamic change of lineage sequence before and after depleting certain unwanted B cells, dynamic change of lineage sequence abundancy during immunization course, lineages which share the same naive B-cell origin between VHH and VH, avoidance of developability liability sequences and a combination thereof.
  • hamming distance refers to the number of positions at which the corresponding symbols are different between two sequences of equal length.
  • the term "grouped antibodies by lineage”, “lineage-related antibodies” and “antibodies that related by lineage” as well as grammatically - equivalent variants thereof, are antibodies that are produced by cells that share a common B cell ancestor.
  • Antibodies that are related by lineage bind to the same epitope of an antigen and are typically very similar in sequence, particularly in their light chain and heavy chain CDR3s.
  • Both the heavy chain and light chain CDR3s of lineage - related antibodies can have an identical length and a near identical sequence (i.e., differ by up to 5, i.e., 0, 1, 2, 3, 4 or 5 residues).
  • minimal CDR3 distance of a specific CDR3 is the smallest hamming distance of this CDR3 comparing with all other CDR3 of the same length. In some embodiments, the minimal CDR3 distance is equal to or less than 1.
  • the B cell ancestor contains a genome having a rearranged light chain VIC region and a rearranged heavy chain VDJ region, and produces an antibody that has not yet undergone affinity maturation.
  • "Naive" or "virgin" B cells present in spleen tissue are exemplary B cell common ancestors.
  • Lineage related antibodies is intended to describe a group of antibodies that are produced by cells that arise from the same ancestor B-cell.
  • a “lineage group” contains a group of antibodies that are related to one another by lineage.
  • the term "at least the CDR3s" or “at least the CDR3 sequences” refers to only CDR3 sequences, CDR3 sequences in conjunction with CDR1 and / or CDR2 sequences or a sequences of at least 50 contiguous amino acids of the variable domain, up to the entire length of the variable domain, where the sequence contains a CDR3 sequence.
  • lineage tree refers to a diagram, resulting from a cladistics analysis, which depicts a hypothetical branching sequence of lineages leading to the individual species of interest. The points of branching within a lineage tree are called nodes.
  • constructing a phylogenetic tree refers to the computational act of making a phylogenetic tree from sequences.
  • Lineage is used interchangeably with “group”, and sometimes a group of antibodies related by lineage is referred to as a “lineage group”.
  • group or “lineage” is exclusive, in that a sequence can belong to only one group or lineage.
  • subgroup refers to a further grouping of sequences in a lineage based on unique features or signatures.
  • Subgroup is not exclusive, which means one sequence can be in different subgroups. For example, one sequence can have two, three, four, five, or six unique features at the same time.
  • Subgrouping is only for VHH. Applying VHH sequence signatures can help to select / narrow-down testing lineages
  • lineage analysis refers to the analysis of the theoretical line of descent of an antibody, which is usually done by analyzing a lineage tree.
  • sequence read refers to a sequence of nucleotides determined by a sequencer, which determination is made, for example, by means of base calling software associated with the technique.
  • obtaining the amino acid sequences refers to obtaining a file containing amino acid sequences.
  • a nucleic acid sequence can be translated into an amino acid sequence in silico.
  • the abundance of a protein can be determined by, e.g., counting sequence reads encoding that protein.
  • the protein encoded by the most sequence reads is the most abundant protein.
  • anchor and “anchor binder” as used herein interchangeably, is referred to conventional antibody generated with single B-cells sorting or heterohybridoma having native H and L pairing, with that, ones can "position/pair” heavy chain lineage and light chain lineage which consist of a group of sequences derived from clonal expansion of naive B- cell H and L sequences after encountering the epitope of antigen. Lineages can be "anchored” considering the amino acid sequences of heavy and light chains that are known to pair with one another. In these embodiments, the branches are rotated around their nodes until there is a minimal number of cross - overs (e.g., no crossovers) between the anchored sequences.
  • the leaves that are known to pair can be connected by an edge. If the leaves that are known to pair are connected by an edge, the intervening leaves, in theory, can pair with one another as long as they do not create a cross - over event with an edge or one another.
  • an antibody which binds specifically to an antigen
  • telomere binding refers to the ability of an antibody to preferentially bind to a particular antigen that is present in a homogeneous mixture of different molecules. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable molecules in a sample, in some embodiments more than about 10 to 100 fold or more than e.g., about 1000 - or 10, 000 fold.
  • the term "does not substantially bind" to a protein or cells can mean that it cannot bind or does not bind with a high affinity to the protein or cells, i.e., binds to the protein or cells with an K D of 2xl0 6 M or more, more preferably 1 x 10 5 M or more, more preferably 1 xlO 4 M or more, more preferably lxlO 3 M or more, even more preferably 1 x 10 2 M or more.
  • the term "high affinity" for an IgG antibody can refer to an antibody having a KD of lxlO -6 M or less, preferably lxlO -7 M or less, more preferably lxlO -8 M or less, even more preferably lxlO 9 M or less, even more preferably 1x10 10 M or less for a target antigen.
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a "therapeutically effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmaco-dynamic effect of the treatment.
  • the therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
  • the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
  • a "CDR grafted antibody” is an antibody comprising one or more CDRs derived from an antibody of a particular species or isotype and the framework of another antibody of the same or different species or isotype.
  • a "humanized antibody” has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non human species antibody, when it is administered to a human subject.
  • certain amino acids in the framework and constant regions of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody.
  • the constant region(s) from a human antibody are fused to the variable region(s) of a non-human species.
  • a humanized antibody is a CDR grafted antibody comprising one or more CDRs derived from an antibody of a particular species or isotype and the framework of human antibodies.
  • one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886, 152 and 5,877,293.
  • chimeric antibody refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
  • one or more of the CDRs are derived from a human antibody.
  • all of the CDRs are derived from a human antibody.
  • the CDRs from more than one human antibodies are mixed and matched in a chimeric antibody.
  • a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody. Other combinations are possible.
  • biparatopic antibody refers to an antibody binds to two non overlapping epitopes of an antigen.
  • the biparatopic antibody comprises heavy chain only VHHs without light chain.
  • the biparatopic antibody comprises both heavy chain only VHHs and conventional VHVVL 1 pairs.
  • the biparatopic antibody comprises two conventional VHVVL 1 pairs.
  • the biparatopic antibody has a first heavy chain and a first light chain from a monoclonal antibody targeting one epitope, and an additional antibody heavy chain and light chain targeting another epitope.
  • the additional light chain or heavy chain can be different from the first light or heavy chains.
  • an antibody of the disclosed invention can be assessed using one or more techniques well established in the art.
  • an antibody can be tested by ELISA assays, for example using a recombinant antigen protein.
  • Still other suitable binding assays include but are not limited to a flow cytometry assay in which the antibody is reacted with a cell line that expresses the human antigen, such as HEK293 cells.
  • the binding of the antibody including the binding kinetics (e.g., KD value) can be tested in BIAcore binding assays, Octet Red96 (Pall) and the like.
  • single B-cell sorting refers to the sorting of isolated and separated single B cells based on antigen specificity. Technologies for single-cell separation, isolation, and sorting include but are not limited to: FACS (fluorescent activated cell sorting, e.g. using a fluorescent-tagged antigen to isolate cells that bind the antigen), ISAAC (immunospot array assays on a chip), LCM (laser-capture microdissection), microengraving, and droplet
  • FACS fluorescent activated cell sorting, e.g. using a fluorescent-tagged antigen to isolate cells that bind the antigen
  • ISAAC immunospot array assays on a chip
  • LCM laser-capture microdissection
  • microengraving and droplet
  • a method for generating a camelid heavy chain only antibody or binding portion thereof for recognizing an antigen, particularly for therapeutic applications comprises: [0141] a) Enriching and amplifying antigen-specific B-cells from immunized Camelidae; b) generating antibody NGS libraries comprising VHH 2 , VHH 3 , and VH 1 and VL 1 chain sequences from the antigen-specific B-cells; c) Grouping VHH 2 , VHH 3 , and VH 1 sequence data as phylogenetic lineage clonotypes based on CDR3 - for example by grouping together CDR3 amino acid sequences that differ by 0 or 1 amino acids and have the same length; d) ranking lineages comprising the VHH heavy chain (VHH 2 , VHH 3 ) by lineage priority factors; e) Selecting a representative sequence from lineages of VHH 2 , VHH 3 with top rankings in antibody sequence library according to the lineage priority factors; f) Testing an
  • the antigen comprises a plurality of epitopes.
  • sequences from a plurality of lineages can be selected and tested by repeating step f) and g).
  • f) can include 1) synthesizing DNA of the selected representative sequences, 2) constructing a vector comprising the DNA sequence, 3) expressing the vector in cells, 4) conducting an affinity and bioactivity test against the specific antigen.
  • b) can include 1) Making cDNA from the enriched population of antigen - specific B cells; 2 Sequencing the cDNA to obtain a plurality of VHH 2 , VHH 3 , VH 1 heavy chain sequences and a plurality of VL 1 (VK and VX) light chain sequences to generate a camelid lgG2 (HcAb), lgG3 (HcAb) and IgGl (conventional Ab) library.
  • the generated camelid antibody comprises an lgG2.
  • the generated camelid antibody comprises an lgG3.
  • Another aspect of the invention lies in a method for generating a camelid antibody or binding portion thereof for recognizing an antigen, particularly for therapeutic applications, the method comprises: a) Enriching and amplifying antigen-specific B-cells from immunized Camelidae; b) generating antibody NGS libraries comprising VHH 2 , VHH 3 , VH 1 and VL 1 chain sequences from said antigen-specific B-cells; c) grouping VHH 2 , VHH 3 , VH 1 , and VL 1 sequence data as phylogenetic lineage clonotypes based on CDR3; d) IgGl VHVVL 1 lineage pairing according to an anchor binder generated by single B-cell sorting and heterohybridoma approaches; e) ranking lineages and lineage pairs from step c) and step d) by lineage priority factors; f) selecting a representative sequence from lineages of VHH 2 , VHH 3 and lineage pairs of VHVVL
  • sequences from a plurality of lineages can be selected and tested by repeating step f) and g).
  • g) can include 1) synthesizing DNA encoding the selected representative amino acid sequences, 2) constructing a vector comprising the DNA sequence, 3) expressing the vector in cells, 4) conducting an affinity and bioactivity test against the specific antigen.
  • b) can include 1) Making cDNA from the enriched population of antigen - specific B cells; 2 sequencing the cDNA to obtain a plurality of VHH 2 , VHH 3 , VH 1 heavy chain sequences and a plurality of VL 1 (VK and VX) light chain sequences to generate a camelid lgG2 (HcAb), lgG3 (HcAb) and IgGl (conventional Ab) library.
  • HcAb camelid lgG2
  • HcAb lgG3
  • IgGl conventional Ab
  • the generated camelid antibody comprises an lgG2. In one embodiment, the generated camelid antibody comprises an lgG3. In one embodiment, the generated camelid antibody comprises a conventional IgGl. In one embodiment, the ranking of lineage pairs in step e) is based on lineage priority factors of VH 1 lineages of said lineage pairs. In one embodiment, the ranking of lineage pairs in step e) is based on lineage priority factors of VL 1 lineages of said lineage pairs. The method can further comprise testing to determine if antibody binding to the antigen inhibits binding of the antigen to another protein, e.g. whether antibody binding inhibits a ligand from specifically binding to its cognate receptor.
  • a method for generating humanized camelid antibodies targeting an antigen comprises a) enriching and proliferating antigen-specific B-cells from immunized camelids, b) generating antibody NGS libraries comprising VHH 2 , VHH 3 , and VH 1 chain sequences from antigen-specific B-cells, c) grouping sequences of VHH 2 , VHH 3 , and VH 1 in the NGS library by lineages, d) identifying a substitutable position in a parent VHH antibody or VH 1 sharing the same naive B-cell origin by comparing its amino acid sequence to the amino acid sequences of a number of related antibodies that each bind to the same epitope as the parent antibody in the same lineage, e) substituting amino acids at one or more of the substitutable positions of the parental VHH antibody or VH 1 antibody by correspondingly positioned amino acids in the human antibody; f) testing an antibody comprising the substituted residues within the selected sequences to determine if the antibody binds to
  • substitutable position is in the CDRs regions. In one embodiment, the substitutable position is in the FRs regions.
  • a camelid conventional IgGl antibody generated in this invention can be humanized by substituting amino acids at one or more of the substitutable positions of the parental IgGl antibody by corresponding positioned amino acids in the human antibody.
  • FIG.2 An embodiment of the method for camelids immunization and antigen-specific B cells isolation / proliferation is schematically illustrated in FIG.2; such embodiment comprises:
  • B cells are referred to memory cells, plasmablasts and different stages of B-cells which have cell membrane lgG2 (HcAb), IG3 (HcAb) and IgGl (conventional IgG); 4) enriching antigen-specific B-cells with cell surface antibodies either through physical-surface antigen panning, or magnetic bead isolation or flow sorting; 5) activating the enriched B cells for cells proliferation in the presence of the antigen, camelid CD40-L expression cells and growth factors.
  • the activation step allows one to selectively stimulate memory B-cells to differentiate and become plasma cells, which are rapidly dividing and expressing larger amounts of antibody.
  • immune responses of lgG2, lgG3 and IgGl in antisera are monitored by a) purifying lgG2, lgG3 and IgGl with Protein-A and Protein-G columns at different pH elution conditions; b) analyzing immune response titers of lgG2,3 and IgGl; or c) testing bioactivities of lgG2, 3 and IgGl with desired immunoassays.
  • the activating step of the method which only proliferate B cells that have surface-tethered antibodies that are actually bound to the antigen, has three effects (1) the activating step causes only those B cells that are specifically bound to the antigen to proliferate, thereby increasing the relative concentration of those cells relative to the cells that are non- specifically bound to the support (2) activating step of the method causes the expression of HcAb or conventional IgG heavy and light chain mRNA to be induced only in the B cells that are specifically bound to the antigen (B) these "rare” antigen-specific B cells, but express antibodies with high affinity or specificity or recognizing "rare” epitopes have been amplified to improve signal-to-noise ratio significantly
  • antigens used for enrichment includes, but not limited to:
  • a Complex Immunogen an animal may be immunized with multiple antigens or cells or tissue, or biofluid, and antigen-specific B cells for each of a plurality of antigens may be enriched separately from one another or as a whole. The antigen-specific B cells can then be activated and collected separately from one another or as a whole.
  • the simplicity of VHH offers an advantage for high throughput VHH cloning and expression, and it is relatively easier to identify each corresponding antigen of VHH by deconvoluting a complex immunogen.
  • the complex immunogen can be deconvoluted by methods including but not limited to protein array or immunoprecipitation-based mass spectrometry method or cells, tissue antigen-cDNA library screening method.
  • VHH 2 , VHH 3 , VH 1 , VK and VX NGS libraries can be prepared from B cells expressing lgG2 (HcAb), lgG3 (HcAb) and IgGl (conventional Ab), respectively.
  • cDNA amplification can be done by steps of:
  • VHH 2 , VHH 3 , VH 1 , VK and VX mRNA capture/RT-PCR
  • NGS libraries for VHH 2 , VHH 3 , VH 1 , VK and V can be prepared, for example, by
  • cDNA in NGS libraries can be sequenced by High throughput sequencing of the library, for example using an lllumina MiSeqS00x2 instrument.
  • Sequences can be structured by a bioinformatics process: - quality evaluation using NGSQCTookit, assembly R1/R2 reads, translation and then identifying CDR 1,2,3.
  • VHH 2 , VHH 3 , VH 1 , VK and VX antibodies can be grouped using NGS data of, for example, CDR3 amino acid sequences, to construct phylogenetic lineage clonotypes.
  • a lineage is defined by a group of sequences which are from same naive B cells
  • a lineage can be defined as a group with amino acid sequences in their CDR3 region differing by no more than 1 amino acid (hamming distance is 1 or less, or same CDR3 sequence if total amino acids within 5 aa). It is presumed that the amount of lineages reflects the amount of naive B cells in a library, also the number of epitopes that these antibodies recognize. (FIG. 4).
  • lineage size correlates with antibody maturation and clonal expansion.
  • Bioinformatic methods allow structure and visualization of the data for a rational approach to candidate antibodies selection. For each NGS library, up to 10,000 of lineages are possible to be identified by sequences structuring through a bioinformatic process comprising: QC using NGSQCTookit, assembly R1/R2 reads, translation, identifying CDRsl,2,3 and then lineage grouping based on CDR3 similarity.
  • VHH (VHH 2 , VHH 3 ) sequences can be further grouped (subgrouped) by their unique sequence signatures.
  • VHH Camelids have evolved multiple mechanisms to further diversify VHH B-cell repertoire and enlarge antigen-binding capability. Sequence "signatures” arising from these mechanisms allow one to further group lineages. Such additional criteria for further grouping lineages can reflect subtle different recognitions of antibodies and help to identify epitopes with unique VHH recognition patterns.
  • the signatures of VHH include but are not limited to: [0168] i) FR2 hydrophilic region: for most VHH antibodies, FR2 has unique amino acids substitutions for conventional IgG: 37Phe/Tyr, 44Glu, 45Arg, and 47Gly;
  • VHH uses this region together with long CDR3 to increase the surface area interacting with antigen;
  • VHH lineage groups which share the same naive B cells origin (same V and J assignment) with the conventional IgGl, which indicates the same or similar epitope recognition by both VHH and IgGl;
  • non-classic VHH which have unique sequence signatures: for example, conserved Trpll8 substituted with Argll8 and or lower hydrophobicity profile in FR3;
  • xiii) the number of cysteines in the amino acid sequence
  • xiv) 2-4 amino acid motifs found in CDR regions. The motif is identified from a 3- d structure of a ligand/receptor complex
  • VHH 2 , VHH 3 Humanization of VHHs (VHH 2 , VHH 3 ), VH 1 -VK and VH ⁇ VX can be guided by lineage analysis.
  • the invention provides a method for identifying positions of an antibody that can be modified without significantly reducing the binding activity of the antibody.
  • the method involves identifying a substitutable position in a parent antibody by comparing its amino acid sequence to the amino acid sequences of a number of related antibodies that each bind to the same antigen and epitope as the parent antibody in the same lineage.
  • the amino acid at the substitutable position may be substituted for a different amino acid without significantly affecting the activity of the antibody.
  • the subject methods may be employed to change the amino acid sequence of a CDR without significantly reducing the affinity of the antibody.
  • the invention finds use in a variety of therapeutic and diagnostic applications.
  • Bispecific/biparatopic antibodies or antigen-binding fragments can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmarm, Clin. Exp. Immunol. 79: 315-321 (1990), Kos- telny et al., J. Immunol. 148:1547-1553 (1992).
  • bispecific antibodies can be formed as "diabodies" or "Janusins.”
  • a plurality of VHH variable domains can also be connected by linkers to form bivalent and multivalent antibodies.
  • VH 1 and VL 1 (VK or VX) pairing lineages can be identified by considering VH /L 1
  • anchor amino acid sequences found to pair with one another in antibodies secreted by heterohybridomas and/or flow-sorted single B-cells (illustrated in FIG. 6).
  • a challenge for camelid conventional IgGl development with NGS technology is how to identify original, natural H and L pairs.
  • Two approaches to establish anchor H and L (Kand l) lineages are typically used; (1) heterohybridoma and (2) single B-cell sorting. After lineage pairs are further grouped with these anchors, representative sequence H/L pairs from each lineage pairs are selected for DNA synthesis, binding screening and bioactive tests as VHH antibodies.
  • Camelids fuse lymphocytes with mouse myeloma fusion partner cell lines such as SP/20 and generate heterohybridomas; screen supernatants of heterohybridomas with ELISA and bioactive assays; sequence VH 1 and VL 1 from the selected heterohybridomas; these pairs of VH and VL are used as anchors to pair VH 1 and VL 1 lineages from IgGl NGS library.
  • This method is able to capture the entire antigen-specific B-cell repertoire from an immunization, including HcAb and conventional IgGl with NGS and takes advantage of the simplicity of HcAb:
  • a lineage of antibodies recognizing a broad spectrum of epitopes of the antigen can be selected (illustrated in Fig.7).
  • Each lineage or lineage pair is presumed to recognize one unique epitope, thus one representative sequence (VHH) or one representative pair (VH /L 1 ) from top 100 lineages/lineage-pairs (e. g 70 sequences for VHHs and 30 sequence pairs for VH ⁇ VGI or VH [0203] V0) are selected for gene synthesis, binder screening and bioactive tests.
  • the lineage selection criteria include but not limited to:
  • developability liability sequences there are sequences which consist of some amino acids that can cause developability problems such as thermal stability (hydrophobic core, charge cluster residues and others), chemical stability (deamidation and isomerization), solubility (surface hydrophobicity and others) and heterogeneity (glycosylation) (Tomoyuki Igawa et al. mAbs, 2011). Selection of these lineages or lineage pairs should be avoided.
  • the selection criteria can also be a combination of the above priority factors.
  • the selected lineage sequences or pair is used for DNA synthesis and constructed into expression vectors such as VHH, scFv, Fab, HcAb, Camelidae IgGl and human Fc chimeric.
  • expression vectors such as VHH, scFv, Fab, HcAb, Camelidae IgGl and human Fc chimeric.
  • both selected VHH lineage and selected VH /L 1 lineage pair could share the same naive B-cell origin
  • more pairs in the same top ranking lineage pairs in the first round of selection e.g ., 70 sequences for VHHs and 30 sequence pairs for VH 1 -VK or VH ⁇ VX are selected for gene synthesis, binder screening and bioactive tests because representative sequence pairs from VHM/K or VH ⁇ VX need more combinational tests before the optimal pairs are identified.
  • More sequences and pairs in the next 100 top ranking lineages are selected for gene synthesis, binder screening and bioactive tests if the first 100 antibodies don't produce desired results.
  • the CDR3 sequence is the main determinant for binding to an epitope while CDR1 and CDR2 are more or less involved in determining other binding properties.
  • the leads e.g. marked by @ and + in FIG. 8
  • This step also helps to build-up a big candidate pool for further antibody drug development.
  • the selected sequences or pairs are used for DNA synthesis; and constructed into expression vectors such as VHH, scFv, Fab, HcAb, Camelidae IgGl and human Fc chimeric.
  • More sequences and pairs can be selected until the best antibody is identified, and the remaining clones kept as a pool of further candidates.
  • VHHs VHH 2 and VHH 3
  • VH /K VH ⁇ VX
  • Non-classic VHH genes sharing the same naive cells with conventional VH which is helpful to (1) subgroup VHH lineages (2) select HcAb and conventional IgG recognizing the same or similar epitopes (3) facilitate humanization for both HcAb and conventional IgG.
  • VHH domains typically display a high sequence identity with human type 3 VH domains (VH3), likely accounting for their low immunogenicity (Cortez-Retamozo V, Int J Cancer. 98(3):456-62, 2002).
  • camelids VH 1 , VX and VK domains of conventional antibodies also reveal significant homologous to their human counterparts in both sequence and structure (Alex Klarenbeek, et al., mAbs 7:4, 693--706; 2015).
  • FIG. 9 As we know, sequences within the same lineage group share the same or similar CDR3 sequence and recognize the same epitope.
  • VHH (without FR2 hydrophilic amino acids) are derived from the same locus of IGHV3 or IGHV4, D and J genes as conventional VH 1 , the lineages structures between VHH and VH 1 sequences in these groups are similar and can further support humanization design each other through lineage analysis.
  • the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition of the invention can comprise a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or that have complementary activities.
  • Example 1 Identification of a group of VHH antibodies specifically binding to an antigen using the B cell isolation and amplification (BIA)/NGS sequence analysis and Single B- Cell methods
  • Cell line TIB-181 (EL4.IL-2) was obtained from the American Type Culture Collection and stably transfected with a pCMV-6-based vector comprising a cDNA encoding human CD40L and that confers expression of human CD40L. A stable cell line was selected and treated with mitomycin as feeder cells.
  • splenocytes are isolated for alpaca conditioned medium preparation.
  • Activation medium containing 10% FBS, phytohemagglutinin (PHA) and phorbol myristate acetate (PMA) is prepared.
  • PHA phytohemagglutinin
  • PMA phorbol myristate acetate
  • 4x10 s splenocytes are suspended in [0237] T175 flask in activation medium and incubated at 37°C 5% CC ⁇ for 48h. After incubation, the supernatant is collected and filtered as alpaca conditioned medium.
  • Antigen for example, 400pg Keyhole Limpet Cyanin ("KLH) in 0.5mL PBS is emulsified with 0.5mL complete Freund’s adjuvant.
  • KLH Keyhole Limpet Cyanin
  • the emulsified antigen is subcutaneously injected along the neck and the back of an alpaca. Five injections of ⁇ 200 pL (or less) are performed. Immunizations are performed 3 times in 14 day intervals
  • EDTA-containing blood samples from an immunized alpaca are diluted twofold with lx DPBS containing 2% FBS. Then the diluted blood is slowly put on a Ficoll-Paque PLUS density gradient media for density centrifugation. The upper layer is drawn off and the lymphocyte layer is transferred to a clean centrifuge tube. The PBMCs are then washed twice with lx DPBS.
  • the spleen from an immunized alpaca is washed with lx DPBS and put in a clean dish. Balloon the spleen by injecting the medium until most lymphocytes are released. Then use the bottom of a 20cc syringe to crush the spleen into pieces. Use of a lot of force when crushing helps get the best possible yield of lymphocytes. All of the released cells are collected by gentle centrifugation, e.g. at 1400rpm. The
  • lymphocytes isolation from the lymph nodes mesenteric lymph nodes and inguinal lymph nodes are collected from an immunized alpaca. Lymphocytes are released by grinding the Lymph nodes in RPMI 1640 medium. The cells are passed through a cell mesh and collected by centrifugation. 5x the volume of the pellet of Red Cell Lysis Buffer is added to the sample and the sample is allowed to stand for at least 4 min to remove RBC. Then RPMI 1640 is added to terminate the red cell lysis. The lymphocytes are then washed twice with lx DPBS.
  • the tibia and radius of an immunized alpaca are opened at both ends of the bone and the bone marrow is taken.
  • Cells are released by grinding the bone marrow in RPMI 1640 medium.
  • the cells are passed through a cell mesh and collected by centrifugation. 5x the volume of the pellet of Red Cell Lysis Buffer is added and the sample is allowed to stand for at least 4 min to remove RBC.
  • RPMI 1640 medium is added to terminate the lysis and the lymphocytes are washed twice with lx DPBS.
  • Penicillin-Streptomycin and 0.05 mM 2-Mercaptoethanol to get a 1 M/mL cell suspension.
  • the cells are preincubated for lh at 37°C, in a 6 well culture plate for non-specific adhesion of macrophage and monocytes to the plate surface. After pre-incubation, unbound cells are collected and counted.
  • Penicillin-Streptomycin to get a 1 million/mL cell suspension.
  • the cells are incubated for 1.5 h at 37°C, at 5 million per 10cm dish pre-coated with antigen for specific B cell panning. After incubation, the plated cells are washed with RPMI1640 medium for 2-10 times until only a few cell are found in suspension. All of the unbound cells are collected and counted, the count being designated as Al.
  • B cell medium 20mL B cell medium are added into the dish containing B cells retained by the panning.
  • the B cell medium should contain 10% FBS, 1% Penicillin-Streptomycin, 10% alpaca conditioned medium, various growth factors, for example one or more interleukins, at a concentration of 1 to 50 ng/ml and 2.5M MMC pre-treated feeder cells (EL4.IL-2-C3, expressing alpaca CD40L).
  • B cell only and feeder cell only dishes are also cultured as a control in quality control testing. Cells are cultured in 5% CO2 for 8-10 days.
  • BCAF (A2-A3)/(5M-A1)
  • NGS library construction [0255] The B cells from each panning were used to prepare template RNA for NGS cDNA library construction. To calculate sequence enrichment, libraries using B cells before panning were also constructed.
  • Cultured B cells are lysed with TRIZOL Reagent by repetitive pipetting or by passing through a syringe and needle. Use 1 ml of the reagent per 0.5-1 X 10 6 cells. 20% chloroform is added and the tube is agitated for about 15 sec. The aqueous phase is carefully removed using a pipette. An equal volume of isopropanol is added to the aqueous phase and mixed gently. The sample is centrifuged at maximal speed (12,000 rpm) for 10 min. The isopropanol is removed and the pellet is washed with 1 ml 75% ethanol in DEPC treated water and gentle mixing. The pellet is recentrifuged at 7,000 rpm for 1 min. and the RNA is recovered in approximately 70 pi of RNase-free water.
  • RNA-primer mix is heated at 65°C for 5 minutes, and then incubated on ice for at least 1 minute.
  • RT reaction mix is prepared and the reverse transcription is conducted at: 30°C for 10 min; 42°C for 50 min; 75°C for 15 min. iii. cDNA amplification PCR
  • GCAGGTCCCCAAGGTGTCCTGTCC - SEQ ID NO. 64 NGS-leader3_L (GGTGGTCCTGGCTGCTCT - SEQ ID NO. 65), NGS-hingel_L (TTGTGGTTTTGGTGTCTTGGG - SEQ ID NO. 66) and NGS- hinge2_L (GGGGTCTTCGCTGTGGTGCGC - SEQ ID NO. 67) and cycled as follows: 98°C for 3 min; 20 cycles of (98°C for 15 s, 58°C for 30 s and 72°C for 30 s); 72°C for 3 min.
  • the resulting amplicons are purified using TIANGEN ® PCR purification kits with a >300 bp size cutoff according to the manufacturer's instructions.
  • Each amplicon sample is individually barcoded in a second 'tagging' 50 pi PCR reaction containing 25mI 2xPrimer star mix, 18mI RNase Free dH20, ImI of each primer pair (e.g. P5-seqF and P7-indexl-seqR), and lOOng first-round PCR as template, then cycled as follows: 98°C for 30 s; 12 cycles of (98°C for 10 s, 65°C for 30 s and 72°C for 30 s); 72°C for 5 min.
  • the final three amplicons (derived from three different reverse primers ) are pooled and purified from 1.5% (w/v) agarose gels using a TIANGEN ® PCR purification kits.
  • Amplified cDNAs are sequenced using a MiSeq Sequencing System (lllumina,
  • a lineage can be defined by clones having a CDR3 length of at least 12 amino acids, a CDR3 hamming distance (in comparison to a reference sequence) of 0 or 1, and having identical V/J region amino acid sequences.
  • Enrichment scores for sequences or sequence groups are calculated using ratio of frequencies between libraries constructed from B cells before and after panning.
  • Representative clones (20 or more) are selected from different lineages based on lineage priority factors as shown in Figure 7, and the antibodies they produce are tested in various binding assays like ELISA, FACS etc. Clones selected from different lineages are found to typically bind to different epitopes of the target protein. To optimize existing clones, additional clones can be selected from the same lineage as shown in Figure 8. Clones selected from within a lineage are typically shown to bind to different parts of the same epitope
  • Selection A Pick clones with the highest count in a cluster
  • Cluster C328 has 357 fold increase in frequency after enrichment.
  • the top count clone in the cluster, NBL505-A1L1-P3R3_355, is a good choice to select for further tests.
  • Selection B Pick clones with highest enrichment score.
  • an enrichment score can be calculated based on sequence frequency before and after enrichment.
  • the enrichment score can be used to prioritize clone picking.
  • clone NBL505-A1L2-P3R2_5559 is selected based on the Fold Enrichment.
  • PBMCs Peripheral Blood Mononuclear Cells
  • GE Ficoll density gradient centrifugation
  • the cells are incubated with 200pL KLH-biotin diluted to 5pg/mL in MACS buffer(PBS plus 2% FBS plus 2mM EDTA) for 30 min at 4°C and then washed twice with 5ml ice-cold MACS buffer. Then the cells are stained with rabbit anti-llama lgG(FI&L), APC-Streptavidin and live/dead dye.
  • the collected antigen specific alpaca B cells are lysed in the collection tubes, followed by heating to 65 °C for 5min. After cooling to 4 °C, total RNA from the lysed single cell is reverse transcribed in a final volume of 20 pL of 4pL 5xPrimeScipt II Buffer (Takara), 20U RNase Inhibitor (Takara), 200U PrimeScript II RTase(Takara) and 4.5uL RNase Free water (Takara) for 50min at 42 °C, after an initial step of 10 min at 30 °C allowing random hexamers hybridation. The reaction is stopped by incubation for 15 min at 72 °C
  • Variable regions of the rearranged heavy chain (HC) locus, lambda (LCA) or kappa (LCK) light chain loci are next amplified separately from each single cell cDNA by two rounds of nested PCR.
  • the first round of PCR is performed on 3 pL of cDNA at 98 °C for 5min, 98 °C for 15s, 55 °C for lmin for HC (62 °C for LCK and 58 °C for LCA,) and lmin at 72 °C for 40 cycles followed by a final elongation step at 72 °C for 7 min in 2X PrimeStar MAX Buffer (Takara) and lOOnM of primers in a 40-pL reaction volume. 4pL of the first amplification products were further amplified by a second round of PCR.
  • the second round of PCR protocol consists of a denaturation step at 98 °C for 5min and then 40 amplification cycles (30s at 98 °C, 30s at 58 °C for HC, 62 °C for LCK, 58 °C for LCA, and lmin at 72 °C) and a final step at 72 °C for 7 min with 2X PrimeStar MAX Buffer (Takara) and lOOnM of primers in a 50-pL reaction volume.
  • PCR products from each single cell are detected on a 1.5% agarose GelRed gel.
  • PCR products from each well were purified by filtration through a commercially available purification kit (Tiangen).
  • Ligation is performed in a total volume of 20 pL with 10 uL of Genbuilder plus cloning-
  • Ligase (Genscript), 100 ng of digested and purified PCR product and 100 ng of linearized vector.
  • Electrocompetent E. Coli TOP10 bacteria are transformed with 20 pi of the ligation products. Colonies are screened by PCR using PET-SEQ-F (TGCTGGTCTGCTGCTCCTCGC - SEQ ID NO. 68) as the forward primer and PET-SEQ-R as the reverse primer (ACCGTCTATCAGGGCGATGG - SEQ ID NO. 69), respectively.
  • the expected insert band is approximately 700bp in length.
  • MSLN mesothelin
  • this method can also identify clones which are shown to behave either as a blocker or nonblocker for the antigen-ligand complex. For example, among the 7 MSLN binding clones, antibodies from 2 clones are identified which bind to either domain 2 or 3 of MSLN but do not inhibit CA125 binding to MSLN. In contrast, the 5 domain 1 epitope binders are shown to prevent CA125 binding to MSLN.
  • the present disclosed methods are efficiently able to systematically and relationally select antibodies for testing to cover a broad range of epitopes with high-resolution.
  • the isolated PBMCs (viability> 95%) are resuspended in complete RPMI1640 media to get a 10 s /mL cell suspension.
  • 4 mL of the PBMC suspension is added to each well of a 6-well plate and incubated for 1 hour to trap nonspecific binding cells. Then the unbound cells are collected and resuspended to 10 6 /mL in complete RPMI1640 media.
  • 5mL of the cell suspension are added to a 10 cm high-binding petri dish (protein binding capacity > 500 ng/cm 2 ) pre-coated with antigen for 1.5 hour with gentle shaking at 50 rpm, 37 °C.
  • the B cell co-culture medium contains 10% FBS (fetal bovine serum), 1% Penicillin-Streptomycin, 10% alpaca conditioned media from cultures of alpaca blank PBMCs, and various growth factors, for example one or more interleukins, at a concentration of from 1 to 50 ng/ml.
  • FBS fetal bovine serum
  • Penicillin-Streptomycin 10% alpaca conditioned media from cultures of alpaca blank PBMCs
  • various growth factors for example one or more interleukins, at a concentration of from 1 to 50 ng/ml.
  • Random hexamer and CH2-specific primers are used in reverse transcription, respectively.
  • Moass DR Sepulveda J, Pernthaner A, Shoemaker CB. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods. 2007;324(l-2):13-25.
  • VHHs camelid heavy chain antibodies
  • These cDNA are then subjected to two rounds of PCR reactions. All these three libraries were sent to Genscript, Nanjing, China for sequencing on a MiSeq Sequencing System (lllumina, Miseq, 300x2) using a 30% PhiX genomic DNA spike in 4-5 million reads were generated for each sample.
  • Sequences are clustered into lineages/groups as shown in Figure 4, based on same CDR3 length, CDR3 hamming distance smaller or equal to 1 and same mapped V/J germlines. Sequences are further sub-grouped into clusters based on same CDR3 length and CDR3 identity with 80% or more. Over 800 groups were generated for each library. Several lineage priority factors are applied here to select clones from these groups: sequence abundance, classical VHH vs non classical VHH and CDR3 length. 20 clones are selected from 20 different groups and they are
  • Example 2 Discovery of an antibody that blocks PD-1:PD-L1 complexation by epitope prediction
  • FIG. 14 Workflow for discovery of an antibody that blocks a ligand:receptor binding interaction (complexation) is illustrated in Figure 14.
  • the workflow begins with identification of three dimensional structures of the ligand and receptor, and/or of the structure of their complex, by computational or crystallographic methods. Portions of both of the receptor and ligand that form the binding interface can be determined by examination of the interface of the "docked" proteins. Amino acids that interact to establish or stabilize the bound complex can be determined by examination of the structure. Data from experiments that change amino acids at the interface and look at the effect of such a change on the binding strength of the complex can contribute to determining the amino acids that interact to form and stabilize the complex.
  • Short linear amino acid sequence portions of 2 to 4 amino acids of the ligand or of the receptor that lie at the interface of the complex are selected and then an NGS amino acid sequence library is searched to identify antibody cDNA clones that encode the selected short amino acid sequences in a CDR part of the sequence, preferably in a CDR3 portion of the antibody sequence.
  • the screening peptide sequence with 2-4 amino acids length is set as a key word for searching CDR3 sequences from NGS database, and the satisfactory VHH sequences and their abundances are obtained.
  • Sequences identified as present in the NGS library above a selected threshold of abundance are selected for gene synthesis and expressed in HEK293 cells fused with a human lgG4-FC tag.
  • the expressed proteins are purified and then subjected to functional testing, e.g. for antigen binding and inhibition of PD-1:PD-L1 complex formation
  • PYMOL software is used for structure analysis.
  • the structure showed that PD-L1 covered two peptides of PD-1 ( Figure 15), the yellow one is SFVLNWYRMSPSNQTDKLAA (SEQ ID NO. 141), the purple one is YLCGAISLAPKAQIKESLR (SEQ ID NO. 142).
  • the polar contact-residue regions observed in PD-l/PD- L1 crystal structures are selected. Polar contacts between PD-1 and PD-L1 are shown in PYMOL by using 'actions-find-polar contacts-to others excluding solvent'.
  • Figure 15 shows the interface of the PD-1:PD-L1 complex (the data of the complex are publically available - Protein DataBase ID No: 4ZQK), and two adjacent peptides of PD-1 that are potential "blocking" peptides that inhibit complex formation are shown.
  • Figure 16 illustrates results of an interaction analysis between the PD-1 protein and the two adjacent peptides; amino acids that interact between the SFV....SLR peptide of PD-1 and the AFT...RIT peptide of PD-L1 at the interface of their complex are identified by connecting lines.
  • the polar contact residues of PD-L1 were determined, and they are F-D-Q-ADYKR. Because there are very long amino acid intervals (>2 amino acids) in F-D-Q-A, only the peptide ADYKR fits the selection strategy. Two to four amino acid peptides are chosen for VHH selection. Then the peptides ADYK (SEQ ID NO. 145), DYKR (SEQ ID NO. 146), ADY, DYK, YKR, AD, DY, YK and KR are set for screening criteria from NGS database (Table 2).
  • CDR3 sequences including screening peptides were extracted from the NGS database and their abundance was counted to eliminate the repetitive sequences. It is found that 2-4 amino acids are appropriate to search the NGS database; two amino acids might be too short (resulting in an unmanageable number of hits), and four amino acids might restrict the number of choices unduly. Accordingly, 3 amino acid strings were selected to search the NGS database.
  • Nucleic acids encoding the selected VHH antibody sequences are synthesized and inserted into a pCDNA 3.4 vector fused with lgG4-FC tag with a (G4S) 3 linker. The recombinant plasmids are confirmed by sequencing. Then the VHH-FC constructs are transformed in HEK293 cells.
  • Transformed cells are cultured for 5-7 days to obtain the recombinant proteins. Then the recombinant VHH antibodies are purified from filtered culture supernatants. The protein concentration of the obtained antibodies is measured by UV-absorbance at 280 nm. The purity of the purified recombinant antibodies is evaluated by Coomassie-staining of sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and high-performance liquid chromatography (HPLC).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gels
  • HPLC high-performance liquid chromatography
  • VHH-FC proteins are added to the wells and allowed to stand for a period of time, then 1 pg/ml of HRP-conjugated anti-FC antibody is added as detection antibody and the assay reagents are added. The absorbance is read at 450 nm. Binding of nivolumab serves as a positive control and plates coated with BSA are used as the negative control group. [0308] ELISA blocking assay
  • Each well of 96-well microtiter plates is coated with 2 pg/ml of PD-1 protein in PBS and blocked with 1% BSA. Purified VHH-FC protein at a concentration of 5 pg/ml is added to each well.
  • VHH antibodies from the 19 clones are assessed for binding to PD-L1 and for inhibition of formation of a PD-1:PD-L1 complex by ELISA as described above. Results are shown in Tables 8 and 9, respectively:
  • Table 8 PD-L1 binding activity of VHH antibodies expressed from selected cDNA clones
  • 1194-zO-lgG4 is a positive control antibody known to have activity of blocking PD-1:PD-
  • NBL507-BMK2-H4-lgG4 is an irrelevant antibody used as a negative control.
  • Seven of the selected clones produced VHH antibodies that have substantial activity of binding to PD-1, and five of these with the strongest binding were selected for testing for activity of blocking PD-1:PD-L1 complex formation.
  • Clone SS5 is found to exhibit stronger complexation inhibition even than the positive control 1194-zO-lgG4.
  • VHH2 long-hinged
  • VHH3 short- hinged sequences.
  • 6 VHH sequences can be found having either a long region or a short hinge region.
  • an original pool of 249 sequences is found and 135 of them are found to have hinge sequences.
  • 25 nonredundant sequences with long hinge sequences and 89 with short hinge sequences are found.
  • 19 unique CDR3 sequences have long hinge sequences
  • 53 unique CDR3 sequences have short hinge sequences.
  • 6 CDR3 sequences are found to be shared.
  • VHH2/VHH3. 19 single chain antibodies are identified from the 2 animals that show good binding to human BCMA.
  • the 2 VHH sequences shared by long and short hinge pools, named as 1A1 and 1D2 are also shared by the leads from the traditional VH repertoire.
  • the number of sequences shared by the different classes of antibodies are shown in Figure 21A.
  • the 8 VHH CDR3 sequences shared by either a long or a short hinge are shown in Figure 21B, with the highlighted sequences being shared by all VH/VHH2 and VHH3.
  • VH/VHH2/VHH3 are also be found in both of the A1 and A2 animals (see Figure 22). Therefore, picking those sequences which are found in multiple animals/ VH/VHH germlines is considered to be a useful signature of antibodies that exhibit strong, specific binding to their antigen.
  • FR2 hydrophilic region for most of VHH antibodies, FR2 has unique amino acid substitutions compared to conventional IgG: 37Phe/Tyr, 44Glu, 45Arg, and 47Gly.
  • Figure 24 shows the proportion of clones having these FR2 unique amino acids of VHH antibodies in each of three libraries, each against a different antigen (NBL501 (anti-MSLN), NBL504 (anti-PDl) and NBL602 (anti-KLH)).
  • NBL501 anti-MSLN
  • NBL504 anti-PDl
  • NBL602 anti-KLH
  • VHHs have an extra hypervariable region (residues 27-30, according to Kabat's numbering) next to CDR1.
  • VHH antibodies with this region together with a long CDR3 region have increased the surface area interacting with antigen. However, antibodies having this signature are not often seen.
  • the CDR2 domain is normally 5-9 amino acids in length. However, many clones contain a "long CDR2" with a length of 14-17 amino acids. Importantly, VHH containing a long CDR2 are found to have a higher binding affinity for their antigen than those with a shorter CDR2. ELISA data from antibodies from three different libraries are shown in Figure 27.
  • Extra disulfide bond within CDR3 around 5-10% of VHH antibodies have an extra disulfide bond within the CDR3 domain, which may indicate that the epitope bound by the antibody is a more "conformational" recognition site that is formed from the three-dimensional structure of the antigen rather than a short length of a linear amino acid sequence.
  • About 2-19% of CDR3 contain intra-disulfide bonds.
  • Figure 28 shows the proportion in the three libraries of antibodies having an extra disulfide bond within the CDR3 domain (identified as an additional Cys residue - i.e. 2 cysteine amino acids in CDR3).
  • the NBL504 anti-PDl library contains a high percentage antibodies having a long CDR3 domain, and it contains significantly more CDR3 intra disulfide bonds than the other two libraries, which have such clones at about 2%.
  • Extra disulfide bond between CDR1 and CDR2 Although low in frequency, antibodies having an extra disulfide bond between CDR1 and CDR2 can be found, as an extra Cys amino acid CDR1 or CDR2. See, Figures 29 and 30.
  • VHH antibodies have a disulfide bond between CDR1 and CDR3, and in Llama and Alpaca, 74% of VHH antibodies have a disulfide bond between FR2-CDR3. 70-80% of the VHH antibody sequences do not contain additional disulfide bonds. 10-25% of the VHH sequences might contain an additional disulfide bond (4 cysteines in the sequence). Interestingly, 5-10% of the VHH antibody sequences contain an unpaired cysteine. How and if these VHH antibodies can pair with another VHH antibody by forming a disulfide bond between unpaired cysteines is unknown. A total of up to 7 cysteine in a single VHH sequence has been observed. These data suggest that up to 3 intra-domain disulfide bonds can be formed with a remaining unpaired cysteine.
  • Figure 31 shows analysis of the number of cysteine residues in amino acid sequences of selected VHH antibodies in three different libraries.
  • Figure 36, Figure 37 and Figure 38 show that there is a positive correlation between length of the CDR3 doman and antibody affinity assayed by FACS for VHH anti-BCMA antibodies binding to the surface of cells from two different cell lines (CHO.K1 and RPMI8226) expressing BCMA and by ELISA using supernatant. All correlations have p value equal to 0.001 or smaller.
  • CDR3 length is consistently shown to have a significant correlation with binding affinity of the antibody to its antigen. Accordingly it is preferable to select antibodies having a CDR3 domain length longer than 14 amino acids if there are a large number of clones that might be picked.
  • CDR3 variants with a similar length are observed to be likely binding to a similar epitope of a single antigen.
  • antibodies binding to the same epitopes can be identified by lineage-related sequences. For example, clones with CDR3 length difference of 0 or 1 amino acids. Clones 1182, 1202 and 1734 are picked from same lineage of anti-PDl antibodies, and they all compete with Keytruda for PD1 binding (see, Figure 40). It is seen that clones picked from related lineages with a same length of CDR3 are in the same bin. The experiments disclosed herein suggest that lineage-related clones having same length CDR3 domains will have an increased chance of binding to exactly the same epitope. In picking clones based on NGS data it is preferred to reduce the number of such redundant clones in the selected pool.
  • Non-classic VHH which have the same V and J germlines as conventional IgG: VHH lineage groups that share the same naive B-cell origin (same V and J assignment) with conventional IgGl, are indicated as recognizing the same or a similar epitope whether being a VHH antibody or an IgGl antibody.
  • VHH2/VHH3/VH there are 2 out of 81 sequences that are shared by VHH2/VHH3/VH, about 2.5% of the clones.
  • Trp 118 can be found in each of the NBL501, NBL504 and NBL602 libraries, with a percentage up to 3%. See, Figure 41.
  • Convergent motif or sequence signature different animals in the same experimental group could convergently generate the same motif or sequence signature through the same VDJ assignment. Paratope coded by these motifs or sequence signatures may target the functional epitopes. If multiple animals are immunized, pick convergent sequences shared between animals.
  • FR2 hydrophilic region for most of VHH antibodies, FR2 has unique amino acids substitutions for conventional IgG: 37Phe/Tyr, 44Glu, 45Arg, and 47Gly/Leu/Phe.
  • Non-classic VHH which have unique sequence signatures: conserved Trpll8 substituted with Argll8 and or lower hydrophobicity profile in FR4
  • Some alpaca has VHH with long CDR2 (17 aa instead of 8/9), clones with such long CDR2 appear to have high binging affinity.
  • CDR3 length has positive correlation with binding affinity, pick long CDR3 clusters with priority
  • Convergent motif or sequence signature different animals in the same experimental group could convergently generate the same motif or sequence signature through the same VDJ assignment. Paratope coded by these motifs or sequence signatures may target the functional epitopes.
  • Novel canonical binding loop structure the hypermutation hotspots residing at key- sites to determine the canonical loop structures create an interesting potential to diversify the VHH structural repertoire. Crystallographic studies accentuate that the CDR1 and CDR2 loops of camel VHHs often deviate from the known canonical structures of the conventional VH. Sequence-based new Ag-binding loop conformation prediction should support further grouping the lineage (Laura S. Mitchell, Lucy J. Colwell,
  • Daley LP Kutzler MA, et al. Effector functions of camelid heavy-chain antibodies in immunity to West Nile virus. Clin. Vaccine Immunol. 17:239-46, 2010.
  • McCoy LE et al. Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization. J. Exp. Med. 2012.

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Abstract

L'invention concerne un procédé de génération d'une pluralité d'anticorps de camélidés divers pour couvrir des épitopes fonctionnels de la cible avec une haute résolution. L'invention concerne également un procédé de génération d'anticorps de camélidé.
PCT/US2020/020248 2019-02-27 2020-02-28 Procédé à haut rendement basé sur une séquence générant des anticorps de camélidé pour couvrir de larges épitopes avec une haute résolution WO2020176815A2 (fr)

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JP2021549905A JP2022541697A (ja) 2019-02-27 2020-02-28 高解像度で広範なエピトープを網羅するために、ラクダ科動物抗体を生成する配列ベースのハイスループット法
EP20763951.9A EP3930754A4 (fr) 2019-02-27 2020-02-28 Procédé à haut rendement basé sur une séquence générant des anticorps de camélidé pour couvrir de larges épitopes avec une haute résolution
US17/434,722 US20230002757A1 (en) 2019-02-27 2020-08-28 Sequence-Based High Throughput Method Generating Camelids Antibodies to Cover Broad Epitopes with High-Resolution

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WO2020176815A3 (fr) * 2019-02-27 2021-03-11 Zhejiang Nanomab Technology Center Co. Ltd. Procédé à haut rendement basé sur une séquence générant des anticorps de camélidé pour couvrir de larges épitopes avec une haute résolution
WO2021130535A2 (fr) 2019-12-28 2021-07-01 Shanghai Cell Therapy Group Company Co., Ltd. Cellule exprimant des molécules modulatrices immunitaires et système d'expression de molécules modulatrices immunitaires
EP4144898A1 (fr) * 2021-09-07 2023-03-08 New/era/mabs GmbH Procédé de sélection ou de dépistage des anticorps à partir d'une bibliothèque d'anticorps
WO2024094097A1 (fr) * 2022-11-02 2024-05-10 Zhejiang Nanomab Technology Center Co. Ltd. Apprentissage automatique pour une découverte d'anticorps et ses utilisations
WO2024094095A1 (fr) * 2022-11-02 2024-05-10 Zhejiang Nanomab Technology Center Co. Ltd. Découverte d'anticorps par traçage de lignée longitudinale

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WO2023178845A1 (fr) * 2022-03-21 2023-09-28 上海恒润达生生物科技股份有限公司 Procédé de purification de lymphocytes t et son utilisation
WO2024094096A1 (fr) * 2022-11-02 2024-05-10 Zhejiang Nanomab Technology Center Co. Ltd. Sélection de nanocorps à l'aide de caractéristiques de séquence

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EP1976882A2 (fr) * 2006-01-24 2008-10-08 Domantis Limited Ligands qui lient il-4 et/ou il-13
GB2461546B (en) * 2008-07-02 2010-07-07 Argen X Bv Antigen binding polypeptides
US8293483B2 (en) * 2009-09-11 2012-10-23 Epitomics, Inc. Method for identifying lineage-related antibodies
US10465186B2 (en) * 2013-08-19 2019-11-05 Epitomics, Inc. Antibody identification by lineage analysis
EP3161166A4 (fr) * 2014-06-25 2017-11-29 The Rockefeller University Compositions et procédés pour la production rapide de répertoires polyvalents de nanocorps
US10934524B2 (en) * 2015-03-18 2021-03-02 Epitomics, Inc. High throughput monoclonal antibody generation by B cell panning and proliferation
EP3283512A4 (fr) * 2015-04-17 2018-10-03 Distributed Bio Inc Procédé pour l'humanisation de masse d'anticorps non humains
JP2022541697A (ja) * 2019-02-27 2022-09-27 ジャージャン ナノマブ テクノロジー センター カンパニー リミテッド 高解像度で広範なエピトープを網羅するために、ラクダ科動物抗体を生成する配列ベースのハイスループット法

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WO2020176815A3 (fr) * 2019-02-27 2021-03-11 Zhejiang Nanomab Technology Center Co. Ltd. Procédé à haut rendement basé sur une séquence générant des anticorps de camélidé pour couvrir de larges épitopes avec une haute résolution
WO2021130535A2 (fr) 2019-12-28 2021-07-01 Shanghai Cell Therapy Group Company Co., Ltd. Cellule exprimant des molécules modulatrices immunitaires et système d'expression de molécules modulatrices immunitaires
EP4144898A1 (fr) * 2021-09-07 2023-03-08 New/era/mabs GmbH Procédé de sélection ou de dépistage des anticorps à partir d'une bibliothèque d'anticorps
WO2023036852A1 (fr) * 2021-09-07 2023-03-16 new/era/mabs GmbH Procédé de sélection ou de criblage d'anticorps et d'antigènes à partir d'une banque d'anticorps
WO2024094097A1 (fr) * 2022-11-02 2024-05-10 Zhejiang Nanomab Technology Center Co. Ltd. Apprentissage automatique pour une découverte d'anticorps et ses utilisations
WO2024094095A1 (fr) * 2022-11-02 2024-05-10 Zhejiang Nanomab Technology Center Co. Ltd. Découverte d'anticorps par traçage de lignée longitudinale

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